Spirochetes of the bacterial species group Borrelia burgdorferi sensu lato (Bbsl) are obligate parasites transmitted by hard-body ticks and agents of Lyme disease, the most common vector-borne illness in the US and Europe. At least 20 Bbsl species have been discovered worldwide and a similar number of distinct strains coexist in US endemic areas. While Bbsl species and strains vary in human pathogenicity, disease manifestations, ecological invasiveness, and geographic range, genomic variations associated with human-virulent Bbsl strains remain obscure. Here we propose to (1) sequence, assemble, annotate, and disseminate 30+ complete (with whole plasmid complement) Bbsl genomes of newly identified species and strains from the US and across the world; (2) identify genomic variations associated with the most prevalent human-pathogenic Bbsl species and strains through evolutionary analysis of adaptive genomic changes during species divergence and within-population differentiation; and (3) develop a novel, DNA sequencing-based molecular diagnostic technique for co- identification of Bbsl strains and host species from single Ixodes scapularis ticks collected from sites in eastern Long Island (New York State) where Lyme disease is heavily endemic. Previously, the PI and his collaborators have sequenced the full genomes of 23 Bbsl strains, covering the majority of species diversity in the world and strain diversity in the US known at the time. We performed a series of comparative analyses of these genomes and discovered that Bbsl genomes have a stable gene composition, that recombination among coexisting strains fuels its rapid adaptation and host virulence, and that immune escape plays a dominant role in generating strain diversity within its natural populations. We developed and maintains BorreliaBase.org, a web portal for wide dissemination of genome data and results of comparative analyses. The new sequencing initiative will more than double the number and the phylogenetic coverage of high-quality, complete Bbsl genomes in the US and the world. Comparative analysis of these and existing genomes will provide unprecedented statistical power for resolving genetic mechanisms associated with species and strains highly virulent to humans. The new molecular diagnostic techniques would provide precise information on the host species composition for nymph and adult ticks, as well as the most competent reservoir host species for Bbsl in a Lyme disease focal point, improving disease risk prediction and control in local municipalities.